Multiparameter flow cytometric analysis of the effects of indomethacin on adipocyte differentiation in A31T6 cells

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Abstract
A31T6 proadipocytes, derived from BALB/c-3T3 clone A31, develop responsiveness to differentiation-promoting agents at density-arrest and differentiate into adipocytes, as determined by the accumulation of cytoplasmic lipid droplets. A flow cytometric assay is being employed to monitor the acquisition of aspects of the differentiated phenotype. In this study, the assay is used to monitor both the rate of differentiation, as defined by the appearance of cells containing lipid droplets and the rate of adipocyte maturation, which involves measurement of increases in cytoplasmic lipid in cells already committed to the differentiation programme. Specifically, we show that: 1) treatment with a combination of indomethacin and dexamethasone causes the maximum percentage differentiation in the population, 2) addition of indomethacin in combination with either dexamethasone or insulin increases the rate of differentiation, and 3) indomethacin selectively increases the maturation of adipocytes, measured as an increase in the amount of lipid per cell. The cytometric assay used in these experiments has allowed determination of the effects of indomethacin on aspects of the adipocyte phenotype that cannot be measured by standard techniques.